Plasma Triglyceride Levels In Mice Research Articles

Carboxyl-terminal sequences in APOA5 are important for suppressing ANGPTL3/8 activity

Apolipoprotein AV (APOA5) lowers plasma triglyceride (TG) levels by binding to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppressing its capacity to inhibit lipoprotein lipase (LPL) catalytic activity and its ability to detach LPL from binding sites within capillaries. However, the sequences in APOA5 that are required for suppressing ANGPTL3/8 activity have never been defined. A clue to the identity of those sequences was the presence of severe hypertriglyceridemia in two patients harboring an APOA5 mutation that truncates APOA5 by 35 residues ("APOA5Δ35"). We found that wild-type (WT) human APOA5, but not APOA5Δ35, suppressed ANGPTL3/8's ability to inhibit LPL catalytic activity. To pursue that finding, we prepared a mutant mouse APOA5 protein lacking 40 C-terminal amino acids ("APOA5Δ40"). Mouse WT-APOA5, but not APOA5Δ40, suppressed ANGPTL3/8's capacity to inhibit LPL catalytic activity and sharply reduced plasma TG levels in mice. WT-APOA5, but not APOA5Δ40, increased intracapillary LPL levels and reduced plasma TG levels in Apoa5-/- mice (where TG levels are high and intravascular LPL levels are low). Also, WT-APOA5, but not APOA5Δ40, blocked the ability of ANGPTL3/8 to detach LPL from cultured cells. Finally, an antibody against a synthetic peptide corresponding to the last 26 amino acids of mouse APOA5 reduced intracapillary LPL levels and increased plasma TG levels in WT mice. We conclude that C-terminal sequences in APOA5 are crucial for suppressing ANGPTL3/8 activity in vitro and for regulating intracapillary LPL levels and plasma TG levels in vivo.

ANGPTL4 silencing via antisense oligonucleotides reduces plasma triglycerides and glucose in mice without causing lymphadenopathy

Angiopoietin-like 4 (ANGPTL4) is an important regulator of plasma triglyceride (TG) levels and an attractive pharmacological target for lowering plasma lipids and reducing cardiovascular risk. Here, we aimed to study the efficacy and safety of silencing ANGPTL4 in the livers of mice using hepatocyte-targeting GalNAc-conjugated antisense oligonucleotides (ASOs). Compared with injections with negative control ASO, four injections of two different doses of ANGPTL4 ASO over 2 weeks markedly downregulated ANGPTL4 levels in liver and adipose tissue, which was associated with significantly higher adipose LPL activity and lower plasma TGs in fed and fasted mice, as well as lower plasma glucose levels in fed mice. In separate experiments, injection of two different doses of ANGPTL4 ASO over 20 weeks of high-fat feeding reduced hepatic and adipose ANGPTL4 levels but did not trigger mesenteric lymphadenopathy, an acute phase response, chylous ascites, or any other pathological phenotypes. Compared with mice injected with negative control ASO, mice injected with ANGPTL4 ASO showed reduced food intake, reduced weight gain, and improved glucose tolerance. In addition, they exhibited lower plasma TGs, total cholesterol, LDL-C, glucose, serum amyloid A, and liver TG levels. By contrast, no significant difference in plasma alanine aminotransferase activity was observed. Overall, these data suggest that ASOs targeting ANGPTL4 effectively reduce plasma TG levels in mice without raising major safety concerns.

Up-regulation of hepatic CD36 by increased corticosterone/cortisol levels via GR leads to lipid accumulation in liver and hypertriglyceridaemia during pregnancy.

Plasma triglyceride (TG) levels increase as gestation proceeds, and abnormal elevation of TG increases the risk of pregnancy complications. The current study explored the mechanisms involved in hypertriglyceridaemia during pregnancy. Lipid profile and expression levels of key genes involved in liver TG metabolism in non-pregnant and pregnant mice were studied. The effects of pregnancy-related hormones on key genes and the underlying mechanisms were uncovered in vitro and in vivo. Plasma and hepatic TG levels were elevated, while hepatic fatty acid translocase (FAT/CD36) was up-regulated in pregnant mice. Corticosterone and cortisol (endogenous glucocorticoids that are elevated during pregnancy), but not oestradiol or progesterone, significantly up-regulated CD36 in hepatocytes, and this was abolished after knockdown of the glucocorticoid receptor (GR) using a siRNA or in the presence of GR antagonists, RU486 and AL082D06. The luciferase reporter gene and chromatin immunoprecipitation assay further revealed that corticosterone/cortisol promoted the direct binding of GR to the CD36 promoter and up-regulated its transcription. Chronic corticosterone exposure induced liver lipid accumulation and increased plasma TG levels in mice, which were attenuated by RU486 via inhibition of the GR-CD36 pathway. Increased corticosterone/cortisol induces liver lipid accumulation and hypertriglyceridaemia during pregnancy by accelerating fatty acid uptake into hepatocytes via activation of GR and its target gene, CD36. Our results may be useful for the prevention of severe hypertriglyceridaemia and associated pregnancy complications.

Immature sword bean pods (Canavalia gladiata) inhibit adipogenesis in C3H10T1/2 cells and mice with high-fat diet-induced obesity.

Sword bean (SB; Canavalia gladiata) is a perennial vine used as a food and medicinal plant in Asia. SB is rich in nutrients, such as flavonoids and urease, and has various functions, including beneficial effects on dysentery, nausea, and hemorrhoids, as well as anti-inflammatory and antioxidant activity. Various plant parts are used; however, little is known about the physiological effects of SB pods (SBP). In this study, the anti-obesity effects of SBP extract were evaluated. To investigate the anti-obesity effects of SBP extract, we confirmed the SBP extract downregulated lipogenesis-related genes and upregulated genes involved in lipolysis and brown adipocyte markers in differentiated C3H10T1/2 adipocytes in vitro. Next, we use a high-fat diet (HFD)-induced obesity mouse model to determine the anti-obesity effects of SBP extract. Treatment with SBP extract significantly reduced adipocytes. The extract decreased the HFD-induced increases in body weight and plasma triglyceride levels in mice after 8 weeks. mRNA and protein levels of the adipogenesis and lipogenesis-related factors CCAAT/enhancer binding protein-β, CCAAT/enhancer binding protein-α, peroxisome proliferator-activated receptor-γ (PPARγ), and their target genes Ap2, SREBP-1c, FAS, and SCD-1 were reduced by SBP extract. In contrast, AMP-activated protein kinase and sirtuin1, involved in the thermogenic catabolism of fat, were activated by SBP extract in adipocytes and white adipose tissue, increasing the expression of peroxisome proliferator-activated receptor gamma coactivator-1α, peroxisome proliferator-activated receptor-α (PPARα), and uncoupling protein 1 and activating thermogenic activity. SBP extract exerts an anti-obesity effect by inhibiting lipogenesis-related factors and activating fat-catabolizing factors; it is, therefore, a promising functional food and natural anti-obesity agent.

Reduced miR-181d level in obesity and its role in lipid metabolism via regulation of ANGPTL3

Obesity impacts the endocrine and metabolic functions of the adipose tissue. There is increasing interest in the role of epigenetic factors in obesity and its impact on diabetes and dyslipidemia. One such substance, miR-181, reduces plasma triglyceride levels in mice by targeting isocitrate dehydrogenase 1. In the other hand, the adipocyte differentiation and lipid regulating hormone angiopoietin-like 3 (ANGPTL3) is a known regulator of circulating apolipoproteins through its inhibition of the lipoprotein lipase activity. We aimed to study the miR-181d expression in the blood and adipose tissue in a cohort of obese and non-obese people, assessing its possible role in obesity. We also aimed to confirm whether miR-181d can bind and regulate ANGPTL3. miR-181d expression levels were investigated in 144 participants, 82 who were non-obese (body mass index [BMI] < 30) and 62 who were obese (BMI > 30). miR-181d levels in plasma and adipose tissue were measured by RT-PCR. Hepatocyte cell cultures were assessed by overexpression and 3′-UTR-luciferase assays for miR-181d binding to its target protein and its effect on the protein. The plasma levels of ANGPTL3 were also measured by ELISA. The miR-181d levels were significantly lower in obese than in non-obese individuals. In vitro analysis confirmed miR-181 binding to and repression of the ANGPTL3 transcript. Obesity leads to alterations in miR-181d expression. Its downregulation in obese humans was inversely correlated with ANGPTL3, a protein involved in adipocyte differentiation and lipid metabolism. miR-181d can be used as an inhibitor of ANGPTL3 to reduce the TG plasma level.

ANGPTL8 requires ANGPTL3 to inhibit lipoprotein lipase and plasma triglyceride clearance

Angiopoietin-like (ANGPTL)3 and ANGPTL8 are secreted proteins and inhibitors of LPL-mediated plasma triglyceride (TG) clearance. It is unclear how these two ANGPTL proteins interact to regulate LPL activity. ANGPTL3 inhibits LPL activity and increases serum TG independent of ANGPTL8. These effects are reversed with an ANGPTL3 blocking antibody. Here, we show that ANGPTL8, although it possesses a functional inhibitory motif, is inactive by itself and requires ANGPTL3 expression to inhibit LPL and increase plasma TG. Using a mutated form of ANGPTL3 that lacks LPL inhibitory activity, we demonstrate that ANGPTL3 activity is not required for its ability to activate ANGPTL8. Moreover, coexpression of ANGPTL3 and ANGPTL8 leads to a far more efficacious increase in TG in mice than ANGPTL3 alone, suggesting the major inhibitory activity of this complex derives from ANGPTL8. An antibody to the C terminus of ANGPTL8 reversed LPL inhibition by ANGPTL8 in the presence of ANGPTL3. The antibody did not disrupt the ANGPTL8:ANGPTL3 complex, but came in close proximity to the LPL inhibitory motif in the N terminus of ANGPTL8. Collectively, these data show that ANGPTL8 has a functional LPL inhibitory motif, but only inhibits LPL and increases plasma TG levels in mice in the presence of ANGPTL3.

Inhibition of Pancreatic Lipase and Triacylglycerol Intestinal Absorption by a Pinhão Coat (Araucaria angustifolia) Extract Rich in Condensed Tannin.

The purpose of the present work was to characterize the possible inhibition of pancreatic lipase by a tannin-rich extract obtained from the pinhão (Araucaria angustifolia seed) coat, based on the previous observation that this preparation inhibits α-amylases. Kinetic measurements of pancreatic lipase revealed that the pinhão coat tannin is an effective inhibitor. Inhibition was of the parabolic non-competitive type. The inhibition constants, and , were equal to 332.7 ± 146.1 μg/mL and 321.2 ± 93.0 μg/mL, respectively, corresponding roughly to the inhibitor concentration producing 50% inhibition ([I]50). Consistently, the pinhão coat extract was also effective at diminishing the plasma triglyceride levels in mice after an olive oil load; 50% diminution of the area under the plasma concentration versus the time curve occurred at a dose of 250 mg/kg. This observation is most probably the consequence of an indirect inhibition of triglyceride absorption via inhibition of pancreatic lipase. For the pinhão coat tannin, this is the second report of a biological activity, the first one being a similar inhibition of the absorption of glucose derived from starch as a consequence of an inhibitory action on α-amylases. Taken together, these effects represent a potential anti-obesity action, as suggested for other polyphenol or tannin-rich preparations.

Lipolysis, and not hepatic lipogenesis, is the primary modulator of triglyceride levels in streptozotocin-induced diabetic mice.

Diabetic hypertriglyceridemia is thought to be primarily driven by increased hepatic de novo lipogenesis. However, experiments in animal models indicated that insulin deficiency should decrease hepatic de novo lipogenesis and reduce plasma triglyceride levels. To address the discrepancy between human data and genetically altered mouse models, we investigated whether insulin-deficient diabetic mice had triglyceride changes that resemble those in diabetic humans. Streptozotocin-induced insulin deficiency increased plasma triglyceride levels in mice. Contrary to the mouse models with impaired hepatic insulin receptor signaling, insulin deficiency did not reduce hepatic triglyceride secretion and de novo lipogenesis-related gene expression. Diabetic mice had a marked decrease in postprandial triglycerides clearance, which was associated with decreased lipoprotein lipase and peroxisome proliferator-activated receptor α mRNA levels in peripheral tissues and decreased lipoprotein lipase activity in skeletal muscle, heart, and brown adipose tissue. Diabetic heterozygous lipoprotein lipase knockout mice had markedly elevated fasting plasma triglyceride levels and prolonged postprandial triglycerides clearance. Insulin deficiency causes hypertriglyceridemia by decreasing peripheral lipolysis and not by an increase in hepatic triglycerides production and secretion.

A role of apolipoprotein D in triglyceride metabolism

Apolipoproteins (apo) are constituents of lipoproteins crucial for lipid homeostasis. Aberrant expression of apolipoproteins is associated with metabolic abnormalities. Here we characterized apolipoprotein D (apoD) in triglyceride metabolism. Unlike canonical apolipoproteins that are mainly produced in the liver, apoD is an atypical apolipoprotein with broad tissue distribution. We show that circulating apoD is present mainly in HDL and, to a lesser extent, in LDL and VLDL and that its plasma levels were reduced in db/db mice with visceral obesity and altered lipid metabolism. Elevated apoD production, derived from adenovirus-mediated gene transfer, resulted in significant reduction in plasma triglyceride levels in mice. This effect was attributable to en-hanced LPL activity and improved catabolism of triglyceride-rich particles. In contrast, VLDL triglyceride production remained unchanged in response to elevated apoD production. These findings were recapitulated in high-fat-induced obese mice. Obese mice with elevated apoD production exhibited significantly improved triglyceride profiles, correlating with increased plasma LPL activity and enhanced postprandial fat tolerance. ApoD was shown to promote LPL-mediated hydrolysis of VLDL in vitro, correlating with its TG-lowering action in vivo. Apolipoprotein D plays a significant role in lipid metabolism. These data provide important clues to clinical observations that genetic variants of apoD are associated with abnormal lipid metabolism and increased risk of metabolic syndrome.

Heparan Sulfate 2-O-Sulfotransferase Is Required for Triglyceride-rich Lipoprotein Clearance

Hepatic clearance of triglyceride-rich lipoproteins depends on heparan sulfate and low density lipoprotein receptors expressed on the basal membrane of hepatocytes. Binding and uptake of the lipoproteins by way of heparan sulfate depends on the degree of sulfation of the chains based on accumulation of plasma triglycerides and delayed clearance of triglyceride-rich lipoproteins in mice bearing a hepatocyte-specific alteration of N-acetylglucosamine (GlcNAc) N-deacetylase-N-sulfotransferase 1 (Ndst1) (MacArthur, J. M., Bishop, J. R., Stanford, K. I., Wang, L., Bensadoun, A., Witztum, J. L., and Esko, J. D. (2007) J. Clin. Invest. 117, 153-164). Inactivation of Ndst1 led to decreased overall sulfation of heparan sulfate due to coupling of uronyl 2-O-sulfation and glucosaminyl 6-O-sulfation to initial N-deacetylation and N-sulfation of GlcNAc residues. To determine whether lipoprotein clearance depends on 2-O-and 6-O-sulfation, we evaluated plasma triglyceride levels in mice containing loxP-flanked conditional alleles of uronyl 2-O-sulfotransferase (Hs2st(f/f)) and glucosaminyl 6-O-sulfotransferase-1 (Hs6st1(f/f)) and the bacterial Cre recombinase expressed in hepatocytes from the rat albumin (Alb) promoter. We show that Hs2st(f/f)AlbCre(+) mice accumulated plasma triglycerides and exhibited delayed clearance of intestinally derived chylomicrons and injected human very low density lipoproteins to the same extent as observed in Ndst1(f/f)AlbCre(+) mice. In contrast, Hs6st1(f/f)AlbCre(+) mice did not exhibit any changes in plasma triglycerides. Chemically modified heparins lacking N-sulfate and 2-O-sulfate groups did not block very low density lipoprotein binding and uptake in isolated hepatocytes, whereas heparin lacking 6-O-sulfate groups was as active as unaltered heparin. Our findings show that plasma lipoprotein clearance depends on specific subclasses of sulfate groups and not on overall charge of the chains.

Water Extract of Houttuynia cordata Thunb. Leaves Exerts Anti-Obesity Effects by Inhibiting Fatty Acid and Glycerol Absorption

Houttuynia cordata Thunb. is used in folk medicine for diuresis and detoxification. However, it has not yet been reported to have an anti-obesity effect. We found that the water extract of H. cordata leaves (WEH) inhibited the corn oil-induced increase in plasma triglyceride levels in mice. WEH also inhibited the oleic acid- and glycerol-induced increase in the levels of plasma nonesterified fatty acids and glycerol, respectively. Moreover, WEH had anti-obesity effects in mice with high-fat-diet-induced obesity. Therefore, WEH may be able to prevent or reduce obesity induced by a high-fat diet.

A Highly Conserved Motif within the NH2-terminal Coiled-coil Domain of Angiopoietin-like Protein 4 Confers Its Inhibitory Effects on Lipoprotein Lipase by Disrupting the Enzyme Dimerization

Lipoprotein lipase (LPL) is a principal enzyme responsible for the clearance of chylomicrons and very low density lipoproteins from the bloodstream. Two members of the Angptl (angiopoietin-like protein) family, namely Angptl3 and Angptl4, have been shown to inhibit LPL activity in vitro and in vivo. Here, we further investigated the structural basis underlying the LPL inhibition by Angptl3 and Angptl4. By multiple sequence alignment analysis, we have identified a highly conserved 12-amino acid consensus motif that is present within the coiled-coil domain (CCD) of both Angptl3 and Angptl4, but not other members of the Angptl family. Substitution of the three polar amino acid residues (His(46), Gln(50), and Gln(53)) within this motif with alanine abolishes the inhibitory effect of Angptl4 on LPL in vitro and also abrogates the ability of Angptl4 to elevate plasma triglyceride levels in mice. The CCD of Angptl4 interacts with LPL and converts the catalytically active dimers of LPL to its inactive monomers, whereas the mutant protein with the three polar amino acids being replaced by alanine loses such a property. Furthermore, a synthetic peptide consisting of the 12-amino acid consensus motif is sufficient to inhibit LPL activity, although the potency is much lower than the recombinant CCD of Angptl4. In summary, our data suggest that the 12-amino acid consensus motif within the CCD of Angptl4, especially the three polar residues within this motif, is responsible for its interaction with and inhibition of LPL by blocking the enzyme dimerization.

Overexpression of apolipoprotein A5 in mice is not protective against body weight gain and aberrant glucose homeostasis

Apolipoprotein A5 (APOA5) is expressed primarily in the liver and modulates plasma triglyceride levels in mice and humans. Mice overexpressing APOA5 exhibit reduced plasma triglyceride levels. Because there is a tight association between plasma triglyceride concentration and traits of the metabolic syndrome, we used transgenic mice overexpressing human APOA5 to test the concept that these mice would be protected from diet-induced obesity and insulin resistance. Male and female transgenic and wild-type mice on the FVB/N genetic background were fed standard rodent chow or a diet rich in fat and sucrose for 18 weeks, during which time clinical phenotypes associated with obesity and glucose homeostasis were measured. We found that APOA5 transgenic (A5tg) mice were resistant to diet-induced changes in plasma triglyceride but not total cholesterol levels. Body weights were similar between the genotypes for females and males, although male A5tg mice showed a modest but significant increase in the relative size of inguinal fat pads. Although male A5tg mice showed a significantly increased ratio of plasma glucose to insulin, profiles of glucose clearance as evaluated after injections of glucose or insulin failed to reveal any differences between genotypes. Overall, our data showed that there was no advantage to responses to diet-induced obesity with chronic reduction of plasma triglyceride levels as mediated by overexpression of APOA5.

Efficient lowering of triglyceride levels in mice by human apoAV protein variants associated with hypertriglyceridemia

Variation in the apolipoprotein A5 (APOA5) gene has consistently been associated with increased plasma triglyceride (TG) levels in epidemiological studies. In vivo functionality of these variations, however, has thus far not been tested. Using adenoviral over-expression, we evaluated plasma expression levels and TG-lowering efficacies of wild-type human apoAV, two human apoAV variants associated with increased TG (S19W, G185C) and one variant (Q341H) that is predicted to have altered protein function. Injection of mice with adenovirus encoding wild-type or mutant apoAV resulted in an identical dose-dependent elevation of human apoAV levels in plasma. The increase in apoAV levels resulted in pronounced lowering of plasma TG levels at two viral dosages. Unexpectedly, the TG-lowering efficacy of all three apoAV variants was similar to wild-type apoAV. In addition, no effect on TG-hydrolysis-related plasma parameters (free fatty acids, glycerol and post-heparin lipoprotein lipase activity) was apparent upon expression of all apoAV variants. In conclusion, our data indicate that despite their association with hypertriglyceridemia and/or predicted protein dysfunction, the 19W, 185C and 341H apoAV variants are equally effective in reducing plasma TG levels in mice.

Altered apolipoprotein A-V expression during the acute phase response is independent of plasma triglyceride levels in mice and humans

Plasma triglyceride (TG) levels are altered during the acute phase response (APR). Plasma levels of the recently discovered apolipoprotein A-V (apoA-V) are inversely associated with plasma TG. The aim of this study was to investigate the change of apoA-V plasma levels and hepatic apoA-V expression during the APR in relation to plasma TG. During human APR plasma apoA-V was decreased as were plasma TG (each P < 0.01). Also early in the course of the murine APR plasma apoA-V levels and hepatic apoA-V expression were decreased and changed in the same direction as plasma TG. Treatment of HepG2 cells with TNF-α and IL-1β decreased apoA-V mRNA levels early by 42% and 55%, respectively (each P < 0.001). However, in promoter/reporter assays the human apoA-V promoter was unresponsive to proinflammatory cytokines. Instead, we demonstrate that a significant decrease in apoA-V mRNA stability in response to treatment with TNF-α and IL-1β is the underlying basis of decreased apoA-V expression during the APR ( P < 0.05). These data demonstrate that (i) apoA-V expression decreases early during the APR due to changes in mRNA stability, and (ii) during the APR apoA-V is not inversely related to plasma TG levels in mice and humans, thereby identifying a relevant pathophysiological setting, in which the previously reported close inverse association between these parameters does not hold true.

Oligomerization state-dependent hyperlipidemic effect of angiopoietin-like protein 4

Angiopoietin-like protein 4 (Angptl4) is the second member of the angiopoietin-like family of proteins previously shown to increase plasma triglyceride (TG) levels in vivo. We recently reported that Angptl4 is a variable-sized oligomer formed by intermolecular disulfide bonds and undergoes regulated proteolytic processing upon secretion. We now show that adenoviral overexpression of Angptl4 potently increases plasma TG levels by a mechanism independent of food intake or hepatic VLDL secretion. We determined that cysteine residues at positions 76 and 80 of Angptl4, conserved among mouse, rat, and human, are required to form higher order structures. By generating adenoviral expression vectors of Angptl4 containing different epitope tags at both N and C termini, we show that loss of oligomerization results in decreased stability of the N-terminal coiled-coil domain of Angptl4 as well as decreased ability to increase plasma TG levels, suggesting that intermolecular disulfide bond formation plays important roles in determining the magnitude of the hyperlipidemic effect of Angptl4. Because Angptl4 is more potent than Angptl3 in increasing plasma TG levels in mice, inappropriate oligomerization of Angptl4 could be associated with disorders of lipid metabolism in vivo.

Protein Region Important for Regulation of Lipid Metabolism in Angiopoietin-like 3 (ANGPTL3): ANGPTL3 IS CLEAVED AND ACTIVATED IN VIVO

Angiopoietin-like 3 (ANGPTL3) is a secreted protein that is mainly expressed in the liver and regulates lipid metabolism by inhibiting the lipolysis of triglyceriderich lipoproteins. Using deletion mutants of human ANGPTL3, we demonstrated that the N-terminal coiled-coil domain-containing fragment-(17-207) and not the C-terminal fibrinogen-like domain-containing fragment-(207-460) increased the plasma triglyceride levels in mice. We also found that the N-terminal region 17-165 was required to increase plasma triglyceride levels in mice and that a substitution of basic amino acid residues in the region 61-66 of the fragment showed no increase in the plasma triglyceride levels and no inhibition of lipolysis by lipoprotein lipase. In addition, when we analyzed ANGPTL3 in human plasma, we detected cleaved fragments of ANGPTL3. By analyzing recombinant ANGPTL3 in mouse plasma, we found that it was cleaved at two sites, Arg221 downward arrow Ala222 and Arg224 downward arrow Thr225, which are located in the linker region between the coiled-coil domain and the fibrinogen-like domain. Furthermore, a cleavage-resistant mutant of ANGPTL3 was determined to be less active than wild-type ANGPTL3 in increasing mouse plasma triglyceride levels but not in inhibiting lipoprotein lipase activity. These findings suggest that the cleavage of ANGPTL3 is important for the activation of ANGPTL3 in vivo.

Direct and Indirect Mechanisms for Regulation of Fatty Acid Synthase Gene Expression by Liver X Receptors

The nuclear receptors LXRalpha and LXRbeta have been implicated in the control of lipogenesis and cholesterol homeostasis. Ligand activation of these receptors in vivo induces expression of the LXR target gene SREBP-1c and increases plasma triglyceride levels. Expression of fatty acid synthase (FAS), a central enzyme in de novo lipogenesis and an established target of the SREBP-1 pathway, is also induced by LXR ligands. The effects of LXR ligands on FAS expression have been proposed to be entirely secondary to the induction of SREBP-1c. We demonstrate here that LXRs regulate FAS expression through direct interaction with the FAS promoter as well as through activation of SREBP-1c expression. Induction of FAS expression in HepG2 cells by LXR ligands is reduced, but not abolished, under conditions where SREBP processing is suppressed. Moreover, LXR ligands induce FAS expression in CHO-7 cells without altering expression of SREBP-1. We demonstrate that in addition to tandem SREBP sites, the FAS promoter contains a high affinity binding site for the LXR/RXR heterodimer that is conserved in diverse animal species including birds, rodents, and humans. The LXR and SREBP binding sites independently confer LXR responsiveness on the FAS promoter, and maximal induction requires both transcription factors. Transient elevation of plasma triglyceride levels in mice treated with a synthetic LXR agonist correlates with transient induction of hepatic FAS expression. These results indicate that the LXR signaling pathway modulates FAS expression through distinct but complementary mechanisms and suggest that the FAS gene may be a critical target in the control of lipogenesis by LXRs.

Farnesoid X-Activated Receptor Induces Apolipoprotein C-II Transcription: a Molecular Mechanism Linking Plasma Triglyceride Levels to Bile Acids

The farnesoid X-activated receptor (FXR; NR1H4), a member of the nuclear hormone receptor superfamily, induces gene expression in response to several bile acids, including chenodeoxycholic acid. Here we used suppression subtractive hybridization to identify apolipoprotein C-II (apoC-II) as an FXR target gene. Retroviral expression of FXR in HepG2 cells results in induction of the mRNA encoding apoC-II in response to several FXR ligands. EMSAs demonstrate that recombinant FXR and RXR bind to two FXR response elements that are contained within two important distal enhancer elements (hepatic control regions) that lie 11 kb and 22 kb upstream of the transcription start site of the apoC-II gene. A luciferase reporter gene containing the hepatic control region or two copies of the wild-type FXR response element was activated when FXR-containing cells were treated with FXR ligands. In addition, we report that hepatic expression of both apoC-II and phospholipid transfer protein mRNAs increases when mice are fed diets supplemented with cholic acid, an FXR ligand, and this induction is attenuated in FXR null mice. Finally, we observed decreased plasma triglyceride levels in mice fed cholic acid- containing diets. These results identify a mechanism whereby FXR and its ligands lower plasma triglyceride levels. These findings may have important implications in the clinical management of hyperlipidemias.